Clinical and microbiological characteristics and follow-up of invasive Listeria monocytogenes infection among hospitalized patients: real-world experience of 16 years from Hungary

Purpose Invasive Listeria monocytogenes infection is rare, but can lead to life-threatening complications among high-risk patients. Our aim was to assess characteristics and follow-up of adults hospitalized with invasive L. monocytogenes infection. Methods A retrospective observational cohort study was conducted at a national referral center between 2004 and 2019. Patients with proven invasive listeriosis, defined by the European Centre for Disease Prevention and Control criteria, were included. Data collection and follow-up were performed using the hospital electronic system, up until the last documented visit. The primary outcome was in-hospital all-cause mortality, secondary outcomes included residual neurological symptoms, brain abscess occurrence, and requirement for intensive care unit (ICU) admission. Results Altogether, 63 cases were identified (57.1% male, median age 58.8 ± 21.7 years), and 28/63 developed a complicated disease course (44.4%). At diagnosis, 38/63 (60.3%) presented with sepsis, 54/63 (85.7%) had central nervous system involvement, while 9/63 (14.3%) presented with isolated bacteremia. Frequent clinical symptoms included fever (53/63, 84.1%), altered mental state (49/63, 77.8%), with immunocompromised conditions apparent in 56/63 (88.9%). L. monocytogenes was isolated from blood (37/54, 68.5%) and cerebrospinal fluid (48/55, 87.3%), showing in vitro full susceptibility to ampicillin and meropenem (100% each), gentamicin (86.0%) and trimethoprim/sulfamethoxazole (97.7%). In-hospital all-cause mortality was 17/63 (27.0%), and ICU admission was required in 28/63 (44.4%). At discharge, residual neurological deficits (11/46, 23.9%) and brain abscess formation (6/46, 13.0%) were common. Conclusion Among hospitalized adult patients with comorbidities, invasive L. monocytogenes infections are associated with high mortality and neurological complications during follow-up. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-024-03478-z.


Introduction
Human listeriosis is a zoonotic infection mainly caused by Listeria monocytogenes (abbreviated as L. monocytogenes), a facultative intracellular Gram positive rod widely spread in the environment due to its high resistance to external exposures, such as low temperature [1].As a result, L. monocytogenes poses a significant hazard in the food industry as human infection is primarily caused by ingesting soil-contaminated vegetables, fruits, water, dairy products, or animal meat [2][3][4][5][6].Asymptomatic carriage of L. monocytogenes can occur in 0.8-3.4%,whichmay also contribute to disease transmission [7].In healthy adults, ingestion of 1.0 × 10 9 bacteria could lead to moderate to severe acute gastroenteritis, while in individuals with predisposing factors, even a lower bacterial concentration (1.0 × 10 2 -10 4 ) could result in severe and invasive infections such as sepsis, meningitis, miscarriage or perinatal infections [1,[8][9][10].The diagnosis of invasive listeriosis is established through microbiological examination by culturing or nucleic acid amplification of relevant clinical samples (e.g.blood, cerebrospinal fluid), and the first-line treatment typically includes ampicillin or penicillin-G, with or without gentamicin [11][12][13][14].
In Hungary, human listeriosis was first identified in 1965, and over the next 25 years, almost half of the 90 documented cases occurred in pregnant or newborn patients, with a mortality rate as high as 45% [15].In the past decade, the European Centre for Disease Prevention and Control has reported 24 to 39 new cases of human listeriosis annually, but no clinical study about invasive listeriosis has been conducted in Hungary so far [8].Therefore, our aim was to assess the clinical and microbiological characteristics of invasive Listeria monocytogenes infections among hospitalized patients during a 16-year period, with a particular emphasis on adverse clinical outcomes and in vitro resistance patterns of isolates.

Study design
We conducted a retrospective observational cohort study of patients hospitalized for proven invasive listeriosis during a 16-year period from January, 2004 to December, 2019 at South Pest Central Hospital, National Institute of Hematology and Infectious Diseases in Budapest, Hungary (SPCH-NIHI).This tertiary referral center has ≥ 100 dedicated beds for infectious diseases.The study protocol adhered to the recommendations of the Helsinki Declaration and national ethical standards, and was approved by the Institutional Review Board of South Pest Central Hospital, National Institute of Hematology and Infectious Diseases in Budapest, Hungary (IKEB/37/2016).This type of study does not require written informed consent from patients according to national regulations.

Patient identification and selection
Patients hospitalized with a probable or proven clinical case during the study period were eligible for inclusion.Patients were identified using the prospective dataarchiving electronic system of the hospital, utilizing 10th International Classification of Diseases codes consistent with admission and discharge diagnoses of listeriosis (A32, A41, P37).To overcome selection bias, a secondary query was performed from the archiving system of the Central Microbiology Laboratory of our center for all Listeria spp.isolated from clinical samples during the study period.All eligible cases were retrospectively evaluated.Included patients were subgrouped based on the development of a complicated disease course, into two subgroups: (I) Uncomplicated disease course and (II) Complicated disease course (see Tables 1, 2 and 3).Complicated disease was defined by the next: (1) in-hospital death or (2) occurrence of L. monocytogenes brain abscess or (3) presence of residual neurological symptoms at discharge.Patients were included if invasive listeriosis was proven microbiologically (see definitions later).Exclusion criteria were as follows: (1) patient data were unavailable in the electronic system, (2) the final diagnosis did not correspond to invasive listeriosis, (3) patient death or transfer to another hospital within ≤ 72 h of admission.Using the database, follow-up was conducted for all included patients until hospital discharge or the last available outpatient visit (follow-up was completed on June, 2023).

Data collection and definitions
Anonymized data were manually entered into an electronic data collection spreadsheet, standardized by the lead investigator.Collected data included: (1) age and sex, (2) comorbidities and predisposing factors for invasive listeriosis, (3) clinical presentation, (4) microbiological and laboratory examinations, (5) types of empirical and targeted antibiotics and supportive therapy, (6) outcomes.Baseline data were recorded on the day of diagnosis.
Invasive listeriosis was defined according to the case definition of the European Centre for Disease Prevention and Control [16].Microbiological criteria were as follows: 1) presence of Listeria spp.detected through culturing methods or polymerase chain reaction (PCR) in a sample obtained from a physiologically sterile site (blood, cerebrospinal fluid, synovium, pericardial, pleural, or peritoneal fluid, etc.) or non-sterile site (placenta, amniotic fluid, meconium, etc.).Clinical criteria included: (1) meningitis, encephalitis, (2) sepsis and febrile influenza-like syndrome, (3) certain local infections (arthritis, endocarditis, endophthalmitis, abscess of unknown etiology), (4) pregnancy (miscarriage, stillbirth, preterm birth) and neonatal (granulomatosis infantiseptica, skin and mucocutaneous lesions of unknown origin) complications.Isolation and identification of L. monocytogenes were performed using classical culturing and Gram staining, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI Biotyper, Bruker, USA), cerebrospinal fluid (CSF) PCR (BioFire FilmArray Meningitis/Encephalitis Panel, bioMérieux, France), and latex agglutination (Listeria Latex Test, Microgen Bioproducts, UK).In vitro antibiotic susceptibility testing followed the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) current at the time of diagnosis [17].Microbiological diagnostics were conducted at the Central Microbiology Laboratory of our hospital.
We defined adequate empirical antibiotic therapy against L. monocytogenes as: (1) a patient with suspected invasive listeriosis receiving ≥ 1 empirically selectable antibiotic within 24 h of diagnosis, in the appropriate dose and formulation, according to international recommendations, (2) no need for antimicrobial escalation before the final microbiological result was known, or (3) the isolate subsequently detected by culture showed in  vitro antibiotic susceptibility to the chosen agent(s), or if presence of the bacterium was not confirmed by culture, intrinsic antibiotic resistance was not suspected [18].Inadequate empirical antibiotic therapy was considered if ≥ 1 of these criteria were not met.Potential choices for adequate antibiotics against Listeria spp.included amoxicillin, ampicillin, meropenem, linezolid, trimethoprim/ sulfamethoxazole, and vancomycin.Escalation of antibiotic therapy was defined as a change in empirical choice to broaden the spectrum of activity before microbiological results.Targeted antibiotic therapy was defined as continuing or de-escalating empirical therapy based on microbiological results.The American College of Chest Physicians/Society of Critical Care Medicine pediatric and adult Systemic Inflammatory Response Syndrome (SIRS) criteria were used to define sepsis [19].Charlson index was used to define comorbidity burden [20].Longterm systemic corticosteroid therapy was defined as ≥ 3 months of ≥ 15 mg/day of prednisone-equivalent dose of systemic glucocorticoids.Immunocompromised conditions were defined as congenital immunodeficiencies, active oncohematological malignancy, solid organ or hematopoietic stem cell transplantation, ongoing immunotherapy/chemotherapy or immunomodulatory therapy, systemic autoimmune diseases, functional or anatomical asplenia, uncontrolled HIV infection, and end-stage liver cirrhosis.

Clinical outcomes
The primary outcome was in-hospital all-cause mortality.Secondary outcomes were: (1) residual neurological symptoms, (2) occurrence of brain abscess, (3) need for intensive care unit (ICU) admission due to L. monocytogenes infection.All outcomes were assessed at hospital discharge or the last available outpatient visit.

Statistical analysis
Continuous and categorical variables are presented as median ± interquartile range (IQR) with minimummaximum values and absolute numbers of cases (n) and percentage relative frequency (%), respectively.Mann-Whitney U-test and Fisher's exact test were used for

Demographic and baseline characteristics
During the study period, 63 cases of invasive listeriosis were identified.The demographic and baseline patient characteristics are shown in

Epidemiology of invasive listeriosis
Invasive listeriosis is a relatively rare but serious infection.In the USA, it ranks as the third most common fatal foodborne infection after Vibrio vulnificus and Clostridium botulinum [21].Additionally, L. monocytogenes is the fifth most common causative agent of communityacquired acute bacterial meningitis in the USA [22,23].In Spain, a multicentric study of 5696 cases revealed an increasing incidence of listeriosis between 1997 and 2015, with 50% of the affected population being ≥ 65 years of age, 7% being pregnant, and 4% being newborns.Roughly half of the patients had risk factors such as malignancy (23%), diabetes mellitus (17%), and chronic liver disease (13%).The overall mortality rate was 17%, with most of the deceased being from the elderly population (68%) [24].Emphasizing prevention and early diagnosis in populations at risk of invasive disease is of cardinal importance [4][5][6].
The incidence of invasive listeriosis at our center was relatively low, as we confirmed 63 cases during a 16-year period.We mostly identified cases of neurolisteriosis and isolated bacteremia, with neonatal and pregnancyassociated infections being underrepresented, possibly due to frequent referral of these patient groups to other centers.Additionally, empirical treatment without proper    diagnosis might be common during pregnancy, along with an emphasis on avoiding certain foods [25].

Clinical characteristics and diagnosis of invasive listeriosis
Results from a retrospective USA study suggest that the primary predisposing factors for neurolisteriosis are active oncohematological malignancy, post-transplantation status, and long-term corticosteroid therapy.In these patient groups, Listeria sp. was the most prevalent pathogen causing acute bacterial meningitis.Furthermore, chronic excessive alcohol consumption, immunosuppressive therapies, human immunodeficiency virus infection, and other comorbidities (such as diabetes mellitus, autoimmune diseases, and hereditary hemochromatosis) should also be considered, although neurolisteriosis without any known predisposing factor can occur quite frequently (36%) [14].
In our cohort, a predisposing risk factor for listeriosis could be identified in 94%, with old age, antacid therapy, chronic liver disease, diabetes mellitus, and long-term corticosteroid treatment being common.The median age in the cohort was high, and higher Charlson score was associated with complicated disease.Most clinical signs were nonspecific for the final diagnosis, but our data suggest that the absence of fever, and the appearance of focal neurological signs with symptoms of cardiorespiratory instability indicate a more severe disease.Patients with uncomplicated disease course, possibly due to lower likelihood of altered mental status, were more likely to complain of headaches and lower back pain.Cranial imaging and EEG results did not identify any localizations or morphological abnormalities highly predictive of neurolisteriosis.Overally, microbiological positivity from CSF cultures was higher than from blood cultures.While the predominance of neurolisteriosis in our cohort may explain this phenomenon, it should be emphasized that blood cultures also play a significant role in identifying bacterial invasion, as shown by previous studies [14,26].The use of non-culture-based microbiological tests, particularly PCR-based assays, is also likely to facilitate accurate diagnosis.

Therapy and outcomes of invasive listeriosis
The majority of isolated L. monocytogenes strains were found to be highly susceptible to beta-lactams in vitro.Isolates also exhibited full in vitro susceptibility to other antibiotics, such as vancomycin, erythromycin, tetracycline, and chloramphenicol, but their clinical use should probably be avoided due to observed in vivo failures and the potential for plasmid-mediated acquired resistance [27,28].Among recommended antibiotics, gentamicin and trimethoprim/sulfamethoxazole showed a low prevalence for moderate susceptibility or resistance, but these drugs are primarily considered as combination or alternative agents [14].While in vitro susceptibility of L. monocytogenes isolates remains high, increasing acquired resistance to several commonly used antibiotics, such as clindamycin, trimethoprim/sulfamethoxazole, and ciprofloxacin, has been observed in other studies from different geographical areas [27][28][29].Knowledge of these susceptibility patterns is particularly crucial when choosing an empirical treatment regimen for immunocompromised patients.While data from our study might offer reassurance in this aspect, continued active monitoring may be necessary.
Surprisingly, 24% of all patients received empirical antibiotics that were ineffective against Listeria sp., although the rates were similar in the subcohorts.Literature evidence suggests that early inadequate therapy may correlate with higher mortality and a tendency for residual neurological symptoms [30].Although meropenem exhibits excellent in vitro anti-Listeria activity, its clinical use has been associated with increased mortality in some studies [31,32].In our cohort, higher meropenem and lower ampicillin administration rates among patients with a complicated disease may reflect these prior findings, or the tendency of chosing broader spectrum empirical therapy for patients presenting with a more severe disease.Targeted therapy was initiated 3 days after admission on average, with the majority (76%) receiving ampicillin or an ampicillin-gentamicin combination, aligning with literature recommendations [13].Despite a relatively extended duration of antimicrobial treatment (17 ± 8 days), only 8% of cases were switched to oral therapy.In our cohort, one patient underwent successful source control by surgery.While Listeria sp.associated endovascular infections are rare, they can be severe; for instance, 60% of affected patients experienced vascular rupture in a French study [33].As expected, a significantly higher proportion of supportive therapies were needed among patients with more severe outcomes.Historically, dexamethasone therapy, often initiated in suspected acute bacterial meningitis, was believed to be terminated in cases of invasive listeriosis, as its positive effect on clinical outcomes has not been unequivocally demonstrated and, in some studies, it has been associated with increased mortality and delayed cerebral damage [10,13,34].Nonetheless, a recent prospective nationwide cohort study of 162 neuroinvasive cases showed that the adjusted odds ratio of dexamethasone treatment for an unfavourable outcome was 0.4 (95%CI 0.19-0.81),providing an association with an improvement among patients with acute bacterial meningitis caused by L. monocytogenes [35].

Limitations
Due to the retrospective nature of our study, recall and documentational bias is probably inevitable.Also, conducting a multicentric, prospective study would provide more accurate data collection and inclusion of a larger number of patients.We note that the universal definition of sepsis changed during 2016.Despite these limitations, we believe that our study offers a comprehensive overview of the 16-year experience with invasive listeriosis from a tertiary referral center, and could serve as a starting point for future research as the relatively rare occurrence of this infection makes it difficult to study.

Conclusion
In our study, invasive Listeria monocytogenes infections were associated with high mortality and a propensity for prolonged hospitalizations and residual neurological symptoms during follow-up.

Table 1
Demographic and baseline characteristics of patients with invasive Listeria monocytogenes infection in the cohort, grouped by disease

Table 2
Clinical characteristics of patients with invasive Listeria monocytogenes infection in the cohort at diagnosis, grouped by disease course

Table 3
Laboratory, microbiological and imaging characteristics of patients with invasive Listeria monocytogenes infection in the cohort at diagnosis, grouped by disease course Statistical significance was determined at a two-sided p-value < 0.05.Calculations were performed using IBM SPSS Statistics 23.
** Presence of L. monocytogenes was confirmed from CSF in all cases without any other pathogen statistical comparisons.

Table 4
Results of in vitro antibiotic susceptibility testing of isolates from clinical specimens of patients with invasive Listeria monocytogenes infection in the cohort at diagnosis

Table 5
Clinical outcomes of patients treated with invasive Listeria monocytogenes infection and characteristics of their antimicrobial and supportive therapy, by outcome subgroup